PROJECT SUMMARY/ABSTRACT Neurofibromatosis type 1 is an autosomal dominant hereditary cancer syndrome that affects approximately 1:3000 individuals. Patients with NF1 often harbor dozens to hundreds of neurofibromas that range in biological and clinical behavior from benign to malignant. Approximately 10-20% of individuals with NF1 will develop a malignant peripheral nerve sheath tumor (MPNST), thought to arise from the malignant progression of pre-existing neurofibromas. In particular, plexiform neurofibromas are thought to be the precursor lesion for many patients. Currently, there is no way of knowing which individuals and more specifically, which lesions within any one individual are likely to behave in a malignant fashion. While many patients are screened with standard radiographic techniques such as PET/CT to detect the development of malignancies, they are plaqued by issues around specificity, radiation exposure and cost. Effectively identifying individuals in whom malignant progression will arise is of significant clinical importance in the NF1 community. Once diagnosed, MPNST has a five-year survival rate as low as 20%. Total resection with radical surgery remains the only modality with proven survival benefit. Therefore, detecting incipient MPNSTs prior to metastasis and dissemination provides patients with the best opportunity for long-term survival. In Aim 1, we will decipher the genetic landscape of plexiform neurofibromas. We have previously described the genetic landscape of MPNSTs and we will compare the two to identify unique signatures of both tumor types. Not only do we lack effective detection strategies for MPNST, we are also unable to reliably track disease burden in many individuals after the diagnosis has been made. This is in part due to the fact that conventional imaging modalities such as CT or MRI are confounded by the tremendous background of benign tumors that alter the anatomy and obscure subtle changes with the MPNST. In order to obviate this challenge, we will build on work that we have previously published and preliminary data on MPNSTs demonstrating that malignancies shed cell free DNA (ctDNA) into the circulation. The ctDNA can be distinguished from normal cell free DNA by the presence of mutations in the DNA derived from neoplastic cells. We have developed a digital sequencing based approach, coined Safe-SeqS, that can detect and quantify even low levels of ctDNA (as low as 0.01% of total DNA in the plasma). In Aims 2 and 3, we will harness this genetic understanding to develop tools that can readily and non- invasively distinguish and identify malignant peripheral nerve sheath tumors from the background premalignant and benign neurofibromas in individuals with neurofibromatosis 1 (NF1). At the conclusion of the grant, we will have a better biological understanding of the genetic factors controlling malignant progression of plexiform neurofibromas to MPNST and have minimally invasive tools that can be used to diagnose and detect MPNSTs. This has the potential to immediately impact our ability to improve outcomes for individuals with NF1.